A research program is proposed to investigate in mammary cells the tumorigenic consequences of PIK3CA mutations by using a powerful combination of genetic analyses in mouse models and human cells aiming to address mechanistic questions. The PIK3CA gene (encoding the catalytic p110-alpha subunit of PI3-kinase) is one of the most frequently mutated oncogenes in human cancer. In fact, ~30% of breast carcinomas harbor heterozygous non-random (hotspot) gain-of-function PIK3CA missense mutations present in the segments encoding either the helical or the kinase domain of the enzyme (most frequently E545K and H1047R, respectively). Although both of these mutations increase the kinase enzymatic activity, upregulate the downstream AKT pathway, and promote cell transformation upon overexpression, in human tumors they do not act alone and are associated with molecular changes in other genes. In fact, we have found based upon our examination of human tumor biopsies that that each of the hotspots appears to have shared and in some cases distinct collaborators in the oncogenic process. Therefore, instead of overexpression experiments, it is proposed for an unbiased study of common and unique mutational consequences to simulate the situation in human breast tumors by introducing E545K and H1047R mutations into the endogenous gene by a knock-in approach. The oncogenic consequences of activating the mutations in the mammary epithelium will be assessed by generating mouse mutants expressing conditionally each of the two Pik3ca hotspot mutations. Similar knock-in mutants in human mammary cells will also be studied. Genetic relationships with the downstream oncogene Akt1 and the candidate collaborating partners PTEN, HER2/Erbb2 and IGF1R will then be investigated. In parallel, the immediate consequences of the same PIK3CA mutations will be evaluated in pre-malignant mouse mammary epithelium and immortal human mammary epithelial cells, and will be examined for alterations in signaling, gene expression and cell phenotypes, and compared to tumors. PUBLIC HEALTH RELEVANCE: PIK3CA is the most commonly mutated oncogene in human breast cancer, and mutations of PIK3CA have been shown to increase its enzymatic activity. Considering that PIK3CA is an outstanding candidate for cancer therapy, the proposed projects are timely and important for the identification of deregulated oncogenic pathways collaborating with mutated PIK3CA to drive malignant tumor progression. This information on candidate targets for therapeutic intervention would be crucial for the eventual development of strategies aiming to optimize targeted, combinatorial treatment regimens.